Human complement C3-binding protein from streptococcus pneumoniae

ABSTRACT

The present invention relates to the identification and use of a family of human complement C3-degrading proteinases expressed by  S. pneumoniae . The proteinase has a molecular weight of about 24 kD to about 34 kD as determined on a 10% SDS polyacrylamide gel. A preferred proteinase of this invention includes the amino acid sequence of SEQ ID NO: 2.

FIELD OF THE INVENTION

[0001] This invention relates to Streptococcus pneumoniae and inparticular this invention relates to the identification of an S.pneumoniae protein that is capable of degrading human complementprotein. C3.

BACKGROUND OF THE INVENTION

[0002] This application claims the benefit of a provisional application(Ser. No. 60/044,316) filing on Apr. 24,1997 entitled “Human complementC3-degrading proteinase from Streptococcus pneumoniae.”

[0003] Respiratory infection with the bacterium Streptococcus pneumoniae(S. pneumoniae) leads to an estimated 5000,000 cases of pneumonia and47,000 deaths annually. Those persons at highest risk of bacteremicpneumococcal infection are infants under two years of age and theelderly. In these populations, S. pneumoniae is the leading cause ofbacterial pneumonia and meningitis. Moreover, S. pneumoniae is the majorbacterial cause of ear infections in children of all ages. Both childrenand the elderly share defects in the synthesis of protective antibodiesto pneumococcal capsular polysaccharide after either bacterialcolonization, local or systemic infection, or vaccination with purifiedpolysaccharides. S. pneumoniae is the leading cause of invasivebacterial respiratory disease in both adults and children with HIVinfection and produces hematogenous infection in these patients (Connoret al. Current Topics in AIDS1987;1:185-209 and Janoff et al. Ann.Intern. Med. 1992;117(4):314-324).

[0004] Individuals who demonstrate the greatest risk for severeinfection are not able to make antibodies to the current capsularpolysaccharide vaccines. As a result, there are now four conjugatevaccines in clinical trial. Conjugate vaccines consist of pneumococcalcapsular polysaccharides coupled to protein carriers or adjuvants in anattempt to boost the antibody response. However, there are otherpotential problems with conjugate vaccines currently in clinical trials.For example, pneumococcal serotypes that are most prevalent in theUnited States are different from the serotypes that are most common inplaces such as Israel. Western Europe, or Scandinavia Therefore,vaccines that may be useful in one geographic locale may not be usefulin another. The potential need to modify currently available capsularpolysaccharide vaccines or to develop protein conjugates for capsularvaccines to suit geographic serotype variability entails prohibitivefinancial and technical complications. Thus, the search for immunogenic,surface-exposed proteins that are conserved worldwide among a variety ofvirulent serotypes is of prime importance to the prevention ofpneumococcal infection and to the formulation of broadly protectivepneumococcal vaccines. Moreover, the emergence of penicillin andcephalosporin-resistant pneumococci on a worldwide basis makes the needfor effective vaccines even more exigent (Baquero et al. J Antimicrob.Chemother. 1991;28S;31-8).

[0005] Several pneumococcal proteins have been proposed for conjugationto pneumococcal capsular polysaccharide or as single immunogens tostimulate immunity against S. pneumoniae. Surface proteins that arereported to be involved in adhesion of S. pneumoniae to epithelial cellsof the respiratory tract include PsaA, PspC/CBP112, and IgA1 proteinase(Sampson et al. Infect. Immum. 1994;62:319-324, Sheffield et al. Microb.Pathogen. 1992; 13: 261-9, and Wani, et al. Infect. Immun. 1996;64:3967-3974). Antibodies to these adhesins could inhibit binding ofpneumococci to respiratory epithelial cells and thereby reducecolonization. Other cytosolic pneumococcal proteins such as pneumolysin,autolysin, neuraminidase, or hyaluronidase are proposed as vaccineantigens because antibodies could potentially block the toxic effects ofthese proteins in patients infected with S. pneumoniae. However, theseproteins are typically not located on the surface of S. pneumoniae,rather they are secreted or released from the bacterium as the cellslyse and die (Lee et al. Vaccine 1994; 12:875-8 and Berry et al. Infect.Immun. 1994; 62:1101-1108). While use of these cytosolic proteins asimmunogens might ameliorate late consequences of S. pneumoniaeinfection, antibodies to these proteins would neither promotepneumococcal death nor prevent initial or subsequent pneumococcalcolonization.

[0006] A prototypic surface protein that is being tested as apneumococcal vaccine is the pneumococcal surface protein A (PspA). PspAis a heterogeneous protein of about 70-140 kDa. The PspA structureincludes an alpha helix at the amino terminus, followed by aproline-rich sequence, and terminates in a series of 11 choline-bindingrepeats at the carboxy-terminus. Although much information regarding itsstructure is available, PspA is not structurally conserved among avariety of pneumococcal serotypes, and its function is entirely unknown(Yother et al. J Bacteriol. 1992;174:601-9 and Yother J. Bacteriol.1994;176:2976-2985). Studies have confirmed the immunogenicity of PspAin animals (McDaniel et al. Microb. Pathogen. 1994; 17;323-337). Despitethe immunogenicity of PspA, the heterogeneity of PspA, its existence infour structural groups (or clades), and its uncharacterized functioncomplicate its ability to be used as a vaccine antigen.

[0007] In patients who cannot make protective antibodies to thetype-specific polysaccharide capsule, the third component of complement,C3, and the associated proteins of the alternative complement pathwayconstitute the first line of host defense against S. pneumoniaeinfection. Because complement proteins cannot penetrate the rigid cellwall of S. pneumoniae, deposition of opsonic C3b on the pneumococcalsurface is the principal mediator of pneumococcal clearance.Interactions of pneumococci with plasma C3 are known to occur duringpneumococcal bacteremia, when the covalent binding of C3b, theopsonically active fragment of C3, initiates phagocytic recognition andingestion (Johnston et al. J. Exp. Med 1969;129:1275-1290, Hasin HE, JImmunol. 1972; 109:26-31 and Hostetter et al. J Infect. Dis. 1984;150:653-61). C3b deposits on the pneumococcal capsule, as well as on thecell wall. This method for controlling S. pneumoniae infection is fairlyinefficient. Methods for augmenting S. pneumoniae opsonization couldimprove the disease course induced by this organism. There currentlyexists a strong need for methods and therapies to limit S. pneumoniaeinfection.

SUMMARY OF THE INVENTION

[0008] This invention relates to the identification and use of a familyof human complement C3-degrading proteinases expressed by S. pneumoniae.The protein has a molecular weight of about 24 kD to about 34 kD asdetermined on a 10% SDS polyacrylamide gel. The invention includes anumber of proteins isolatable from different C3-degrading strains of S.pneumoniae.

[0009] In one aspect of the invention, the invention relates to anisolated protein comprising at least an 80% sequence identity of SEQ IDNO: 2 and capable of degrading human complement protein C3. In apreferred embodiment, the protein is isolated from S. pneumoniae oralternatively the protein is a recombinant protein. Preferably theprotein binds human complement protein C3. In a preferred embodiment,the protein has a molecular weight as determined on a 10% polyacrylamidegel of between about 24 kDa to about 34 kDa. A preferred protein of thisinvention is an isolated protein including SEQ ID NO: 2.

[0010] The invention also relates to peptides from the C3-degardingproteinase of this invention and preferably peptides of at least 15sequential amino acids from an isolated protein comprising at least an80% sequence identity of SEQ ID NO: 2 and capable of degrading humancomplement protein C3 and more preferably peptides of at least 15sequential amino acids from SEQ ID NO: 2.

[0011] The protein of claim 9, wherein the protein is a recombinantprotein. In another aspect of this invention, the invention relates to apeptide of at least 15 sequential amino acids from SEQ ID NO: 2.

[0012] The protein of this invention can comprise SEQ ID NO: 2, andpreferably has a molecular weight as determined on a 10% polyacrylamidegel of between about 24 kDa to about 34 kDa. Also preferably the proteindegrades human complement protein C3. Preferred protein or polypeptidesof this invention include a protein comprising amino acids 1-50 of SEQID NO: 2 and a nucleic acid fagment comprising nucleic acids 1246 to1863 of FIG. 1A.

[0013] In another aspect of the invention the invention relates to aprotein that degrades human complement protein C3 and wherein nucleicacid encoding the protein hybridizes to SEQ ID NO: 1 under hybridizationconditions of 6×SSC, 5×Denhardt, 0.5% SDS, and 100 μg/ml fragmented anddenatured salmon sperm DNA hybridized overnight at 65° C. and washed in2×SSC, 0.1% SDS one time at room temperature for about 10 minutesfollowed by one time at, 65° C. for about 15 minutes followed by atleast one wash in 0.2×SSC, 0.1% SDS at room temperature for at least 3-5minutes.

[0014] The invention also relates to an immune-system stimulatingcomposition comprising an effective amount of an immunesystem-stimulating peptide or polypeptide comprising at least 15 aminoacids from a protein comprising at least an 80% sequence identity withSEQ ID NO: 2 and capable of degrading human complement protein C3.

[0015] Preferably the protein is isolatable from S. pneumoniae. In oneembodiment, the immune system stimulating composition further comprisesat least one other immune stimulating peptide, polypeptide or proteinfrom S. pneumoniae.

[0016] The invention further relates to an antibody capable ofspecifically binding to a protein comprising at least a 80% sequenceidentity with SEQ ID NO: 2 and capable of degrading human complementprotein C3. In one embodiment, the antibody is a monoclonal antibody andin an other embodiment, the antibody is a polyclonal antibody. Inanother embodiment the antibody is an antibody fragment. The antibody orantibody fragments can be obtained from a mouse, a rat, human or arabbit.

[0017] The invention also relates to a nucleic acid fragment capable ofhybridizing to SEQ ID NO: 1 under hybridization conditions of 6×SSC,5×Denhardt, 0.5% SDS, and 100 μg/ml fragmented and denatured salmonsperm DNA hybridized overnight at 65° C. and washed in 2×SSC, 0.1% SDSone time at room temperature for about 10 minutes followed by one timeat, 65° C. for about 15 minutes followed by at least one wash in0.2×SSC, 0.1% SDS at room temperature for at least 3-5 minutes. In oneembodiment the nucleic acid fragment is isolated from an S. pneumoniaegenome and in another embodiment. the nucleic acid fragment encodes atleast a portion of a protein. In one embodiment, the protein degradeshuman complement C3 and in another embodiment, the nucleic acid fragmentencodes a protein that does not degrade human complement C3.

[0018] In another embodiment, the nucleic acid fragment is in a nucleicacid vector and the vector can be an expression vector capable ofproducing at least a portion of a protein. Cells containing the nucleicacid fragment are also contemplated in this invention. In oneembodiment, the cell is a bacterium or a eukaryotic cell.

[0019] The invention further relates to an isolated nucleic acidfragment comprising the nucleic acid sequencegctcccagtatgcgtactcgtaaggtagagggaagaaaaaaactagctag.

[0020] In another aspect of this invention, the invention relates to amethod for producing an immune response to S. pneumoniae in an animalincluding the steps of: administering a composition comprising atherapeutically effective amount of at least a portion of a protein toan animal, wherein nucleic acid encoding the protein hybridizes to SEQID NO: 1 under hybridization conditions of 6×SSC, 5×Denhardt, 0.5% SDS,and 100 μg/ml fragmented and denatured salmon sperm DNA, hybridizedovernight at 65° C. and washed in 2×SSC, 0.1% SDS one time at roomtemperature for about 10 minutes followed by one time at 65° C. forabout 15 minutes followed by at least one wash in 0.2×SSC, 0.1% SDS atroom temperature for at least 3-5 minutes; and obtaining an immuneresponse to the protein, in one embodiment the immune response is a Bcell response and in another embodiment, the immune response is a T cellresponse. In a preferred embodiment, the composition is a vaccinecomposition. Preferably the at least a portion of the protein is atleast 15 amino acids in length and also preferably the compositionfurther comprises at least one other protein from S. pneumoniae. In oneembodiment, the protein comprises at least 15 amino acids of SEQ ID NO:2.

[0021] In a further embodiment, the invention relates to a bacteriacomprising an insertional mutation, wherein the insertion mutation is ina gene encoding a protein capable of degrading human complement C3. Inone embodiment, the bacteria comprises an insertional duplicationmutation.

[0022] The invention further relates to an isolated protein of about 24kDa to about 34 kDa from Streptococcus pneumoniae that is capable ofbinding to and degrading human complement C3 and to a method forinhibiting Streptococcus pneumoniae-mediated C3 degradation comprisingthe step of: contacting a Streptococcus pneumonia bacterium withantibody capable of binding to a protein with at least 80% amino acidsequence identity to SEQ ID NO: 2. The invention further relates to anisolated nucleic acid fragment comprising the nucleic acid sequence ofSEQ ID NO: 1 and to an RNA fragment transcribed by a is double-strandedDNA sequence comprising SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE FIGURES

[0023]FIG. 1A provides a gene sequence and FIG. 1B provides an aminoacid sequence of a C3 degrading proteinase of this invention.

[0024]FIG. 2 is a diagram of an insertion duplication mutant accordingto this invention.

[0025]FIG. 3 is a diagram of the restriction analysis of an insert froman insertion duplication mutant of this invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention relates to the identification and isolationof a C3 degrading proteinase with a molecular weight of about 29 kDa (±5kDa) on a 10% SDS-PAGE gel (with a predicted size of about 27.5 kDabased on SEQ ID NO: 1) and nucleic acid encoding the C3 degradingproteinase. The protein was originally identified by electrophoresis ofpneumococcal lysates on SDS-PAGE gels impregnated with C3. It has beenobserved that exponentially growing cultures of pneumococci from severalserotypes were able to first degrade the β-chain then degrade the achain of C3 without producing defined C3 cleavage fragments (Angel, etal. J. Infect. Dis. 170:600-608, 1994). This pattern of degradationwithout cleavage differs substantially from other microbial productssuch as the elastase moiety of Pseudomonas aeruginosa and the cysteineproteinase of Entamoeba histolytica. The gene sequence (SEQ ID NO: 1)encoding a C3 degrading protein according to this invention is providedin FIG. 1A and the amino acid sequence (SEQ ID NO: 2) of the protein isprovided in FIG. 1B.

[0027] The term “degrade” is used herein to refer to enzymes that arecapable of cleaving proteins into amino acids, peptides and/orpolypeptide fragments. The proteins of this invention degrade C3 withoutproducing specific cleavage fragments as observed on a polyacrylamidegel.

[0028] A C3-degrading proteinase of about 29 kDa was isolated from alibrary of insertionally interrupted pneumococcal genes by identifyingthose clones that had increased C3 degrading activity as compared towild type S. pneumoniae. There is at least some preference of theC3-degrading proteinases of this invention for C3 in that, for example,the C3-degrading proteinase does not degrade other proteins, such asalbumin, to a large extent. Exemplary methods for performing insertionduplication mutagenesis and for the identification of clones withelevated C3 degrading activity is provided in Example 1.

[0029] A gene encoding a C3-degrading proteinase is contained within aregion that includes four open reading frames and interruption of thethird open reading frame by homologous recombination severely impairedC3 degradation. ORF3 includes about 726 nucleotides and the sequence ofthe translated protein shares no substantial homology with proteinsregistered in either the GenBank or SwissProt databases.

[0030] The full length gene encoding a C3-degrading proteinase of thisinvention was inserted into a gene expression vector for expression inE. coli. Recombinant C3-degrading proteinase was isolated as describedin the examples. Those of ordinary skill in the art recognize that,given a particular gene sequence such as that provided in FIG. 1, thereare a variety of expression vectors that could be used to express thegene. Further, there are a variety of methods known in the art thatcould be used to produce and isolate the recombinant protein of thisinvention and those of ordinary skill in the art also recognize that theC3 degrading assay of this invention will determine whether or not aparticular expression system, in addition to those expression systemsprovided in the examples, is functioning, without requiring undueexperimentation. A variety of molecular and immunological techniques canbe found in basic technique texts such as those of Sambrook et al.(Molecular Cloning, A Laboratory Manual, 1989 Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) and Harlow et al.(Antibodies, A Laboratory Manual. Cold Spring Harbor. N.Y.; Cold Springharbor Laboratory Press, 1988).

[0031] The gene encoding the C3 degrading protein of this invention wasidentified using a plasmid library made with pneumococcal genomic DNAfragments from strain CP1200. Although there are a variety of methodsknown for obtaining a plasmid library; in a preferred strategy, aplasmid library was constructed with Sau 3A digested pneumococcalgenomic DNA fragments (0.5−4.0 kb) from pneumococcal strain CP 1200(obtained from D. A. Morrison, University of Illinois, Champagne-Urbana,Illinois and described in Havarstein LF, et al. Proc. Natl. Acad Sci.(USA) 1995;92:11140-11144) and inserted into the Bam HI site of theintegrative shuttle vector pVA 891 (erm^(r), cm^(r); has origin ofreplication for E. coli). This library was transformed into an E. coliDHLα MCR strain by electroporation. A total of 14000 E. colitransformants were obtained by electroporation. Plasmid extractions ofsome randomly selected E. coli transformants revealed that all of themcontained recombinant plasmids.

[0032] Plasmid library DNA was extracted from the E. coli transformantsand was used to transform the CP 1200 parent pneumococcal strain usinginsertional mutatgenesis homologous recombination.

[0033] The pneumococcal strain CP 1200 cells were made competent using apH shift with HCl procedure in CTM medium. The competent cells werefrozen at −70° C. in small aliquots until needed. Eight thousandpneumococcal transformants were produced using these methods.

[0034] Individual pneumococcal transformants were screened by ELISA fortheir altered phenotypic character based on their ability to degrade C3.Bacterial cultures were incubated with C3 (0.83 μg of C3/ml of culture)for about 2 hrs to about 4 hrs and the amount of undegraded C3 left inthe samples was detected by enzyme linked immunoadsorbent assays (ELISA)using HRP-conjugated goat polyclonal antibody specific to humancomplement C3. The assay was standardized so that wells containingundegraded C3 had an O.D. 490=˜1.0. Wells containing degraded C3 had areduced optical density resulting from their reduced ability to bindanti-C3 antibodies. The optical densities of the mutant and parentstrarns were compared to that of negative controls. The negativecontrols were culture medium containing different concentrations of C3.The percent of C3 degrading activity was determined as a ratio ofoptical density of sample to control. Four mutants (SN3, SN4, SN5 andSN6) were identified with elevated C3 degrading activity (about 2-2.2fold higher activity) as compared with the activity of the about 29 kDaC3-degrading protein from parent strain CP1200. This finding wasconfirmed by Western Blot analysis.

[0035] Total DNA from mutants SN3, SN4, SN5 and SN6 was isolated andused for electroporation into E coli DH5α MCR. Low excision rates ofplasmid DNA from integrated plasmids within the pneumonocci genome canproduce small amounts of free plasmid DNA and this DNA can be recoveredwhen the DNA is transformed into E. coli. This allows furthercharacterization of the plasmid. Retransformation of the plasmid backinto pneumococcus verifies the phenotype of the original mutant.

[0036] Protein samples from the native C3-degrading protein and frommutants SN3, SN4, SN5, SN6 were incubated with C3 and separated on a7.5% SDS-PAGE gel under reducing conditions. C3 degrading activity wasassessed using western blot analysis employing HRP-conjugated antibodyto C3. Mutant SN4 and mutant SN4-4G were used in further experiments.Mutant SN4-4G was identified after CP 1200 was retransformed with therecombinant plasmid pLSN4a rescued from SN4. Both mutant SN4 and mutantSN4-4G almost completely degraded C3 after a 4 hr incubation. While thenative C3 degrading protein degraded C3, after a 4 hr incubation, C3degradation was incomplete as compared with a comparable incubationusing mutants SN4 and mutant SN4-4G.

[0037] The plasmid encoding the protein from mutant SN4 was chosen forfurther investigation. Plasmid pLSN4a (encoding mutant SN4) was used toretransform the wild type CP 1200 strain. This resulted in 48pneumococcal mutants with elevated C3 degrading activity. Digestion withrestriction endonuclease Hind III demonstrated that plasmid pLSN4a wasabout ˜7.8 kb and includedan insert that was about ˜2.3 kb.

[0038] Plasmid pLSN4a was used as a hybridization probe in southernhybridization experiments to verify the presence of the insert inchromosomal DNA samples from the pneumococcal mutants. The resultsconfirmed that the vector with insert (pLSN4a) and also the origin ofthe inserts in the mutants SN3 and SN4 were integrated in thechromosomal DNA. Both mutants SN3 and SN4 consisted of two hybridizingjunction fragments of sizes about ˜2.2 kb and about ˜5.8 kb. Thesefragments were also present in their parent strain CP1200. There weretwo other hybridizing fragments at about ˜42 kb and about ˜3.5 kb andthese two fragments together gave a total of about ˜7.8 kb (pLSN4a is˜7.8 kb). These two bands were also present in the vector with insertsample. Both insert and vector included EcoR I sites and represent therecombinant plasmid. Analysis indicated that a gene duplication hadoccurred in the SN4 mutant strain; therefore, the improvedC3-degradation activity could be attributed to increased C3-degrndingprotein in the SN4 mutants.

[0039] The sequence of about 1 kb of the 2338 bp insert was determinedusing whole pLSN4a plasmid as a template. The remaining sequence (about˜1338 bp) with just insert (PCR product) as a template, was sequenced byICBR, University of Florida. Both complementary strands were sequenced.The results indicated that there were four open reading frames with therelative locations provided in the schematic below: ORF1 ORF2 ORF3 (462bp) (144 bp) (726 bp) ORF4 (358 bp)5′_(——————————————————————————————————) 3”3′_(——————————————————————————————————) 5”

[0040] No significant homology was found between the derived amino acidsequence of the above ORFs and protein sequences from the proteindatabases tested. The ORF3 nucleic acid sequence encoding a C3 degradingproteinase of this invention is provided in FIG. 1A and is designatedSEQ ID. NO 1. The amino acid sequence of this C3 degrading proteinase isprovided in FIG. 1B and is designated SEQ ID NO: 2.

[0041] Out of four opening reading frames (three full and one partial)in the insert, the ORF3 was chosen for further examination because itcontained the largest insert. A 620 bp internal portion (from nucleicacid 1246 to nucleic acid 1863 of FIG. 1A) of ORF3 (PCR product) regionwas ligated into the Hind III site of plasmid pVA 891 and the constructwas transformed into CP 1200 competent cells to knock out the proteinaseactivity. The transformants were tested for their ability to degrade C3after separation on SDS-page gels using western blot analysis. The ORF3disruption mutant had poor activity in comparison with its parent strainCP 1200.

[0042] The entire ORF3 gene (PCR product) was cloned into Nde I and BarnH I sites of pet-28b(+). The vector positions a His-Tag at theN-terminus of the protein. The plasmid construct was transformed into anE coil (DHLα MCR) strain for stabilization before it was trnsformed intoan E. coli (BL 21 DE3) protease deficient strain for protein expression.

[0043] The BL 21 DE3 strain that included the construct (pet 28b(+) withORF3) was induced for ORF3 protein expression. Total cell proteinextracts of the induced and uninduced cultures were tested for C3degrading activity. The expressed His-tagged ORF3 protein was about 29kDa (±5 kDa) on 10% SDS-PAGE gels in the induced samples from theinsoluble protein fraction.

[0044] Solubilition of the ORF3 protein from induced BL21 DE3 cultureswas performed by treating the sample with: a) TES (50 mM, 1 mM, 1 M); b)6 mM G-HCl+1 mM DTT; c) 6 mM G-HCl+1 mM DTT+1% Tween 20; and d) 6 mMG-HCl+1 mM DTT+1% Triton X -100. Both treatments “c” and “d” resulted insoluble protein. Treatment “c” was used to produce solubilizedrecombinant C3 degrading protein that was used for further proteinstudies.

[0045] Guanidine-HCl and DTT were removed from the expressed His-TaggedORF3 protein samples by dialysis. The protein was subjected to Nickelcolumn purification and the eluted His-Tagged protein was visualized ona 10% SDS-PAGE gel.

[0046] The isolated protein encoded by ORF 3 was incubated with humancomplement C3 for 4 hrs at 37° C. in the presence of PBS. Controlsamples without the protein samples were used as negative controls forcomparative purposes. The samples were run on SDS-PAGE gel underreducing conditions as and analyzed for the structure of C3 by WesternBlot assay using polyclonal antibodies to human compiement C3. Theresults indicated that the samples contained a protein encoded by theORF 3 region and that the protein degraded human C3 protein. Both α andβ chains of C3 molecules were susceptible to degradation. In theseexperiments while the α chain was almost completely degraded, the βchain was also degraded, but to a somewhat lesser extent.

[0047] The C3 degrading proteins of this invention were designated CppAproteinases and the genes of this invention are designated CppA. Theproteins of this invention have an apparent molecular weight on a 10%SDS-polyacrylamide gel of about 29 kDa (±5 kDa) and preferably has amolecular weight of about 24 kDa to about 34 kDa As described above,Example 5 indicates that the proteinase is conserved throughout S.pneumoniae strains. However, those of ordinary skill in the art willrecognize that some variability in amino acid sequence is expected andthat this variability should not detract from the scope of thisinvention. For example, conserved mutations do not detract from thisinvention nor do variations in amino acid sequence identity of less thanabout 80% amino acid sequence identity and preferably less than about90% amino acid sequence identity where the protein is capable ofdegrading human complement protein C3, and particularly where theprotein is isolated or originally obtained from an S. pneumoniaebacterium.

[0048] Some nucleic acid sequence variability is expected among thestrains as is some amino acid variability. Conserved amino acidsubstitutions are known in the art and include, for example, amino acidsubstitutions using other members from the same class to which the aminoacid belongs. For example, the nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and tyrosine. The polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine and glutamine. Thepositively charged (basic) amino acids include arginine, lysine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid. Such alterations are not expected to affectapparent molecular weight as determined by polyacrylamide gelelectrophoresis or isoelectric point. Particularly preferredconservative substitutions include, but are not limited to, Lys for Argand vice verse to maintain a positive charge; Glu for Asp and vice versato maintain a negative charge; Ser for Thr so that a free —OH ismaintained: and Gln for Asn to maintain a free NH₂. A preferred proteinof this invention includes a protein with the amino acid sequence of SEQID NO: 2. Other proteins include those degrading human complementprotein C3 and having nucleic acid encoding the protein that hybridizesto SEQ ID NO: 1 under hybridization conditions of 6×SSC, 5×Denhardt,0.5% SDS, and 100 μg/ml fragmented and denatured salmon sperm DNAhybridized overnight at 65° C. and washed in 2×SSC, 0.1% SDS one time atroom temperature for about 10 minutes followed by one time at, 65° C.for about 15 minutes followed by at least one wash in 0.2×SSC, 0.1% SDSat room temperature for at least 3-5 minutes are also contemplated inthis invention. Polypeptides or peptide fragments of the protein canalso be used and a preferred protein of this invention comprises aminoacids 1-50 of SEQ ID NO: 2.

[0049] The proteins of this invention can be isolated or prepared asrecombinant proteins. That is, nucleic acid encoding the protein, or aportion of the protein, can be incorporated into an expression vector orincorporated into a chromosome of a cell to express the protein in thecell. The protein can be purified from a bacterium or another cell,preferably a eukaryotic cell and more preferably an animal cell.Alternatively, the protein can be isolated from a cell expressing theprotein, such as a S. pneunoniae cell. Peptides of the CppA proteinaseare also considered in this invention The peptides are preferably atleast 15 amino acids in length and preferred peptides are peptides withat least 15 sequential amino acids from SEQ ID NO: 2. Another preferredprotein fragment includes amino acids 1-50 of SEQ ID NO: 2.

[0050] Nucleic acid encoding CppA proteinase is also part of thisinvention. SEQ ID NO: 1 is a preferred nucleic acid fragment encoding aCppA proteinase. Those of ordinary skill in the art will recognize thatsome substitution will not alter the CppA proteinase sequence to anextent that the character or nature of the CppA proteinase issubstantially altered. For example, nucleic acid with an identity of atleast 80% to SEQ ID NO: 1 is contemplated in this invention. A methodfor determining whether a particular nucleic acid sequence falls withinthe scope of this invention is to consider whether or not a particularnucleic acid sequence encodes a C3-degrading proteinase and has anucleic acid identity of at least 80% as compared with SEQ ID NO: 1.Other nucleic acid sequences encoding the CppA proteinase includesnucleic acid encoding CppA where the CppA has the same sequence or atleast a 90% sequence identity with SEQ ID NO: 2 but which includesdegeneracy with respect to the nucleic acid sequence. A degenerate codonmeans that a different three letter codon is used to specify the sameamino acid. For example, it is well known in the art that the followingRNA codons (and therefore, the corresponding DNA codons, with a Tsubstituted for a U) can be used interchangeably to code for eachspecific amino acid: Phenylalanine (Phe or F) UUU or UUC Leucine (Leu orL) UUA, UUG, CUU, CUC, CUA or CUG Isoleucine (Ile or I) AUU, AUC or AUAMethionine (Met or M) AUG Valine (Val or V) GUU, GUC, GUA, GUG Serine(Ser or S) UCU, UCC, UCA, UCG, AGU, AGC Proline (Pro or P) CCU, CCC,CCA, CCG Threonine (Thr or T) ACU, ACC, ACA, ACG Alanine (Ala or A) GCU,GCG, GCA, GCC Tyrosine (Tyr or Y) UAU or UAC Histidine (His or H) CAU orCAC Glutamine (Gln or Q) CAA or CAG Asparagine (Asn or N) AAU or AACLysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAU or GACGlutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU or UGCArginine (Arg or R) CGU, CGC, CGA, CGG, AGA, AGC Glycine (Gly or G) GGUor GGC or GGA or GGG Termination codon UAA, UAG or UGA

[0051] Further, a particular DNA sequence can be modified to employ thecodons preferred for a particular cell type. For example, the preferredcodon usage for E. coli is known, as are preferred codons for animalsand humans. These changes are known to those of ordinary skill in theart and therefore these gene sequences are considered part of thisinvention. Other nucleic acid sequences include nucleic acid fragmentsof at least 30 nucleic acids in length from SEQ ID NO: 1 or othernucleic acid fragments of at least 30 nucleic acids in length wherethese fragments hybridize to SEQ ID NO: 1 under hybridization conditionsof 6×SSC, 5×Denhardt, 0.5% SDS, and 100 μg/ml fragmented and denaturedsalmon sperm DNA hybridized overnight at 65° C. and washed in 2×SSC,0.1% SDS one time at room temperature for about 10 minutes followed byone time at, 65° C. for about 15minutes followed by at least one wash in0.2×SSC, 0.1% SDS at room temperature for at least 3-5 minutes.

[0052] The nucleic acid fragments of this invention can encode all, none(i.e., fragments that cannot be transcribed, fragments that includeregulatory portions of the gene, or the like) or a portion of SEQ ID NO:2 and preferably containing a contiguous nucleic acid fragment thatencodes at least nine amino acids from SEQ ID NO: 2. Because nucleicacid fragments encoding a portion of the CppA proteinase arecontemplated in this invention, it will be understood that not all ofthe nucleic acid fragments will encode a protein, polypeptide or peptidewith C3 degrading activity. Further, the nucleic acid of this inventioncan be mutated to remove or otherwise inactivate the C3 degradingactivity of this protein. Therefore, fragments without C3 degradingactivity that meet the hybridization requirements described above arealso contemplated. Methods for mutating or otherwise altering nucleicacid sequences are well described in the art and the production of animmunogenic, but enzymatically inactive protein can be tested fortherapeutic utility. Preferred nucleic acid fragments include gct cccagt atg (claim 34).

[0053] The nucleic acid fragments of this invention can be incorporatedinto nucleic acid vectors or stably incorporated into host genomes toproduce recombinant protein including recombinant chimeric protein. Avariety of nucleic acid vectors are known in the art and include anumber of commercially available expression plasmids or viral vectors.The use of these vectors is well within the scope of what is ordinaryskill in the art. Exemplary vectors are employed in the examples, butshould not be construed as limiting on the scope of this invention.

[0054] This invention also relates to antibody capable of bindingspecifically to a protein of about 29 kDa, and preferably a protein ofabout 24 kDa to about 34 kDa, from S. pneumoniae and preferably wherethe protein is capable of degrading human complement C3. Polyclonalantibody can be prepared to a portion of the protein or to all of theprotein. Similarly, monoclonal antibodies can be prepared to all or to apeptide fragment of the about 29 kDa C3 degrading protein of thisinvention. Methods for preparing antibodies to protein are well knownand well described, for example, by Harlow, et al. (supra). In apreferred example, the antibodies can be human derived, rat derived,mouse derived or rabbit derived. Protein-binding antibody fragments andchimeric fragments are also known and are within the scope of thisinvention.

[0055] The invention also relates to the use of immune stimulatingcompositions. The term “immune stimulating” or “immune systemstimulating” refers to protein or peptide compositions according to thisinvention that activates at least one cell type of the immune system.Preferred activated cells of the immune system include phagocytic cellssuch as macrophages, as well as T cells and B cells. Immune stimulatingcompositions comprising the peptides, polypeptides or proteins of thisinvention can be used to produce antibody in an animal such as a rat,mouse, rabbit, a human or an animal model for studying S. pneumoniaeinfection. Preferred immune stimulating compositions, include an immunestimulating amount of at least a peptide including at least 15 aminoacids from the CppA proteinase. The immune stimulating composition canfurther include other proteins in a pharrnaceutically acceptable buffer,such as PBS or another buffer recognized in the art as suitable and safefor introduction of proteins into a host to stimulate the immune system.The immune stimulating compositions can also include other immune systemstimulating proteins such as adjuvants or immune stimulating proteins orpeptide fragments from S. pneumoniae or other organisrns. For example, acocktail of peptide fragments may be most useful for controlling S.pneumoniae infection. Preferably one or more fragments of the proteinsof this invention are used in a vaccine preparation to protect againstor limit S. pneumoniae colonization or the pathogenic consequences of S.pneumoniae colonization.

[0056] This invention also relates to a method for inhibitingStreptococcus pneumoniae-mediated C3 degradation comprising contacting aStreptococcus pneumonia bacterium with a protein, such as an antibody oranother protein that is capable of binding to an isolated protein ofabout 24 kDa to about 34 kDa from Streptococcus pneumoniae. The proteincapable of binding to an isolated protein of about 24 ka to about 34 kDacan be an antibody or a fragment thereof or the protein can be achimneric protein that includes the antibody binding domain, such as avariable domain, from antibody that is capable of specificallyrecognizing an isolated protein of about 24 kDa to about 34 kDa fromStreptococcus pneumoniae having C3 degrading activity.

[0057] The isolated S. pneumoniae protein of this invention can beisolated and purified and the isolated protein or immunogenic fragmentsthereof can be used to produce antibody. Peptide fragments orpolypeptide fragments of the protein without C3 degrading ability can betested for their ability to limit the effects of S. pneumoniaeinfection. Similarly, the protein of this invention can be modified,such as through mutation to interrupt or inactivate the C3 degradingcapacity of the protein. Isolated protein can be used in assays todetect antibody to S. pneumoniae or as part of a vaccine or amulti-valent or multiple protein or peptide-containing vaccine for S.pneumoniae therapy.

[0058] It is further contemplated that the proteins of this inventioncan be surface expressed on vertebrate cells and used to degrade C3, forexample, where complement deposition (or activation) becomes a problem,such as in xenotinlantation or in complement-mediatedglomerulonephritis. For example, the recombinant protein, or a portionthereof can be incorporated into xenotransplant cells and expressed as asurface protein or as a secreted protein to prevent or minmizecomplement deposition (and/or complement-mediated inflammation).

[0059] All references and publications cited herein are expresslyincorporated by reference into this disclosure. There are a variety ofalternative techniques and procedures available to those of skill in theart which would similarly permit one to successfully perform theintended invention in view of the present disclosure. It will beappreciated by those skilled in the art that while the invention hasbeen described above in connection with particular embodiments andexamples, the invention is not necessarily so limited and that numerousother embodiments, examples, uses, modifications and departures from theembodiments, examples and uses may be made without departing from theinventive scope of this application.

EXAMPLE 1 Generation of Insertional Duplication Mutants and Recovery ofRecombinant Plasmids from Selected Mutants.

[0060] In a preferred example, insertion-duplication mutagenesis wasused to isolate a gene encoding the C3 degrading proteinase fromStreptococcus pneumoniae of this invention. A plasmid library wascreated with 0.5-4.0 kb chromosomal fragments of pneumococcal strain CP1200 (derivative of R×1; Morrison, D. A., et al. J. Bacteriol.,156:281-290,1983) originally obtained from Dr. Morrsion's lab,University of Illinois at Chicago and inserted into the Bam HI shuttlevector pVA 891 (erm^(r), cm^(r) Marcina, F.L. et. al. Gene. 25:1145-150,1983, obtained from Dr. Marcina (Virginia Commonwealth University,Richmond, Va.). pVA891 has resistance markers for erythromycin (erm) andchloramphenicol (cm). The vector has an origin of replication for E.coli, but the origin is non-replicative in Streptococci. Recombinantplasmid can survive when it integrates into the pneumococcal chromosomalDNA by homologous recombination.

[0061]E. coli DH5 α MCR competent cells were made according to theprocedure given in the Bio-Rad Laboratories manual (Richmond, Calif.)and the library was transformed into the competent cells with Bio-RadGene Pulser apparatus (Bio-Rad Laboratories, Richmond, Calif.) byelectroporation.

[0062]E. coli cells were maintained as freezer stocks in small aliquotsat −80° C. in LB broth in the presence of 10% glycerol. The cells weregrown either in LB or TB broth or on LB agar plates containingappropriate antibiotics (erythromycin 200 μg/ml or chloramphenicol 15 or30 μg/ml or kanamycin 30 μg/ml).

[0063] Electroporation was conducted in 0.1 cm cuvette at 1- 2 kV/cmvoltages and a capacitance of 200 Ω. Transformants were selected on LBmedium containing either chloramphenicol (cm, 30 μg/ml) or erythromycin(erm, 300 μg/ml) or combination of erm and cm (200 μg/ml+15 μg/ml).

[0064] A total of 14000 E. coli transformants were obtained from thelibrary. Plasmid extractions and restriction analysis of randomlyselected E. coli transformants revealed the presence of recombinantplasmids.

[0065] Plasmids or recombinant plasmids were extracted from E. colistrains by polyethylene glycol precipitation procedure (Kreig. P. andMelton. D., in Promega Protocols and Applications p. 106,985-86) or amodified alkaline lysis miniprep protocol (Xiang. C., et al.,Biotechniques. 17:30-32, 1994) a modified alkaline extraction procedure(Birnboim H C and J Doly., Nucl. Acids Res. 7:1513-1523, 1979), orCsCl-ethidium bromide gradient method or Qiagen kit (Plasmid midi kit.,Chartsworth, Calif.). Solutions containing DNA were cleaned directlyfrom agarose gels by GeneClean II kit (BIO 101, La Jolla, Calif.) orQiagen kit (DNA extraction from gels., Chartsorth, Calif.). DNA wascleaned by Wizard DNA clean up kit (Promega Corp., Madison, Wis.).Amplified gene products were also cleaned by Wizard PCR clean up kit.(Promega Corp., Madison, Wis.

[0066] The plasmids were transformed into Pneumococcal cells. Thepneumococcal strains were always maintained as freezer stocks in smallaliquots at −80° C., in THB in the presence of 10% glycerol.Pneumococcal cells were grown without shaking in CAT (Morrison, D. A.,et al., 1983, supra) or THB medium (broth or agar). For transformationexperiments, either complete transformation (CTM) broth (Morrison, D.A., et al., 1983) or THB+0.5% Yeast broth (Yother Janet., et al. J.Bacteriol 168:1463-1465, 1986) and for ELISA experiments, SMP, asynthetic medium (see Table 1) were used. Erytliromycin (0.05 μg/ml) wasemployed as a selective antibiotic marker for pneumococcal mutants.TABLE 1 SMP - a synthetic medium SMP solution # 1 (final volume 2liters): NaCl 10.0 g; NH₄Cl 4.0 g; KCl 0.8 g; Na₂HPO₄ 0.24 g; MgSO40.048 g; CaCl₂ 0.020 g; FeSO₄.7H₂O 0.00011 g; Tribase 9.68 g; add d.H₂Oup to 1 liter pH to 7.55 and then add the following amino acids:L-Arginine 400 mg; L-Asparagine 20 mg (monohydrate 22.8 mg); Glycine 240mg; L-Histidine 300 mg; L-Isoleucine 13.10 mg; L-Leucine 13.10 mg;L-Lysine 840 mg; L-methionine 360 mg; (low-methi. 2.6 mg); L-valine11.70 mg; Uracil 2 mg. Make it up to a final volume of 2 liters. SMPsolution 2 (vitamins): Biotin 0.075 mg; Choline 25 mg; Nictinamide 3.0mg; ca pantothenate 12.0 mg; Pyridoxal HCl 3.0 mg; Riboflavin 1.5 mg;Thiamine 3.0 mg; L-Cysteine HCl 0.5 g; L-Glutamine 0.1 g; Na Pyruvate4.0 g; add water and then make up to 50 ml. Reconstituting SMP: ml Startwith: Solution #1 100 Add: Solution #2 1 Solution #3(25% Glucose) 1.6Solution # 4(4% BSA) 2

[0067] Pneumococcal strain CP 1200 cells were made competent by“competence induction by pH shift” (procedure obtained from Dr.Morrison's lab, Univ, of Illinois at Chicago, Ill.) in CTM medium andthe competent cells were frozen at −70° C. in small aliquots untilrequired. In this procecure, to 125 ml of CTM added 1.20 ml of 1 M HCl(final concentration 9 mM) and 4 ml of 0.2 O.D. (550 nm) of frozenpneumococcal stock cells. This-culture was incubated at 370° C. and O.D.readings of the culture were taken at 20 minute intervals beginningafter 3 hrs of incubation. When the culture reached an O.D. of 0.156(550 mn), 1.2 ml of IN NaOH was added at 370° C. After mixing theculture gently, 1 ml of culture was removed as a ‘0’ time point sample,mixed with 100 μl of glycerol and kept on a prechilled metal block.Similarly, ten ml samples were drawn at each time point of 13, 17, 21and 25 min and each sample was added directly to prechilled 1 ml ofglycerol. Each time point sample was frozen in small aliquots at −70° C.Competence was tested for each time point sample by adding 1 μl of DNA(about 250 ng) to 100 μl of cells and incubating at 370° C. for 25 minfor transformation. The transformation culture was diluted and plated onselective medium (erythromycin 0.05 μg/ml). The time point sample thatshowed the highest transformation efficiency was used for futuretransformation experiments. Transformation of the extracted recombinantplasmid library from E. coli transformants into pneumococcal strainCP1200 yielded about 8,000 pneumococcal transformants indicating thatthe plasmid was inserted into the CP1200 chromosome via homologousrecombination.

[0068] Extraction of pneumococcal chromosomal DNA was performed by aslight modification of the method used in the laboratory of Dr. DonaldA. Morrison, University of Illinois at Chicago. Pneumococcal cells weregrown in THB to an O.D. at 550 nm from 0.3-0.4, then rapidly chilled onice and 0.5 M EDTA was added to a final concentration of 10 mM, thecells were spun at 10,000 g for 10 minutes at 4° C., and the pelletswere resuspended in 1:10 volume of cold STE (50 mM Tris-HCl (pH 8.0), 10mM EDTA (pH 8.0), and 0.1 M NaCl). After a second centrifugation, cellswere resuspended in 1/100 volume of cold STE, lysed with 1% TritonX-100, and incubated at 370C for 5-10 minutes for autolysis. After theaddition of 1% SDS, the cells were swirled in water bath at 50-60° C.for 5 min. RNase (100 μg/ml) and proteinase K (50 μg/ml) were addedsequentially with incubations of 2 hours and 1 hours, respectively. Thecells were extracted twice with one volume of phenol/chloroform and oncewith one volume of chloroform and the supernatant was collected forethanol precipitation. The precipitate was washed twice with 70%ethanol, and the pellet was collected and resuspended in TE (10 mMTris-HCl pH 8.0, 1 mM EDTA) or water as required.

[0069] The plasmid library DNA was extracted by polyethylene glycolprecipitation procedure (Kreig. P. and Melton. D. 1985 supra), frompooled E. coli transformants and used to transform CP 1200, the parentpneumococcal strain following the method that was obtained from Dr.Morrison, University of Illinois at Chicago. For pneumococcaltransformation, frozen pneumococcal competent cells were thawed on iceand to 100 μl of these competent cells, 200 ng to 1000 ng of plasmidlibrary was added in a separate eppendorf tube. This tube was incubatedat 37° C. in a water bath for about 25 min to 35 min and the mixture wasdiluted 1/10 in CAT medium and incubated further for about 1-1.7 hrs.Following the fmal incubation, the mixture was plated by overlayprocedure (method was obtained from Dr. Morrison University of Illinoisat Chicago). The overlay procedure involved pouring four differentlayers of agar (THB or CAT) in a small petri dish as follows: a) firstor base layer: 3 ml of agar; b) second or cells' layer: mixture of 1.5ml of agar and 1.5 ml of broth containing required concentration ofbacterial cells; c) third layer: 3 ml of agar; 4) fourth layer or toplayer: 3 ml of agar containing 4× required concentration of antibiotic(erythromycin, at 0.05 μg/ml×4=6 μg/ml ). The plates were incubated at370° C. Individual transformants were transferred by stab inoculation toindividual wells of microtitre plates containing 100 μl of THB anderythromycin (0.05 μg/ml). The recovered transformants in microtitreplates were diluted 1:10 in SMP medium and grown until early log phase,and screened for their ability to degrade C3 by ELISA.

[0070] Spontaneous excision of recombinant plasmids occur in these kindof pneumococcal mutants with low frequency and therefore, chromosomalDNA preparations of these mutants often include low levels of plasmidDNA (Pearce B J., et al., Mol. Microb. 9(5):1037-1050, 1993).Electroporation of E. coli is a highly efficient way of isolating theplasmid constructs in E. coli for further study. Chromosomal DNA (100ng-200 ng in a final volume of 2 μls) from the individual pneumococcalmutants of interest was electroporated into E. coli DH5 α MCR competentcells to obtain E. coli transformants with recombinant plasmids. One ofthe recovered recombinant plasmids (pLSN4a) (see Table 2) was introducedback into wild type CP 1200 pneumococcal strain by transformation. Thetransformant SN4-4G was again evaluated for its C3 degrading activity byELISA.

[0071] DNA fragments were analyzed by horizontal electrophoresis inagarose gels (0.5% to 1.0%) with Tris-borate EDTA (TBE) buffer orTris-acetic acid EDTA (TAE) buffer (Sambrook, J. E. Fritsch and T.Maniatis.1989). One kb ladder from Gibco BRL or Hindi III or HindIII/EcoRI digested lamda DNA from Boehringer Mannheim, was employed as amolecular weight standard.

[0072] Restriction endonucleases, calf intestinal phosphatase, T4 DNAligase, from Gibco BRL Life Technology, Grand Island, N.Y., BoeltringerMannheim, Indianapolis, Ind., Promega Corp., Madison, Wis., BethesdaResearch Laboratories, Gaithersburg, MD., or New England Biolabs., Inc.,Beverly. Mass., were used as described by the manufacturers'instructions.

[0073] DNA fragments were analyzed by Southern hybridization. DNA wastransferred from gels to MSI Magnagraph nylon membranes (MicronSeparations, Inc., Westboro, Mass.) for hybridization and detectionusing Genius nonradioactive DNA labeling and detection kit (BoehringerMannheim Biochemicals, Indianapolis, Ind.) following the instructionsprovided with the kit. Chromosomal or plasmid DNA either from thepneumococcal or E. coli culture was isolated as described in earliersections. About 100 ng-400 ng of each sample was digested with requiredrestriction enzymes and run on 0.7% agarose gel, transblotted ontoMagnagraph-nylon membrane overnight The rest of the procedure wasperformed as instructed by the manufacturer.

[0074] Bacterial strains and plasmids used in this example and theexamples that follow are summarized in Table 2 below. TABLE 2 BacterialStrains and plasmid constructs Plasmids or recovered recombinant Strainor transformant Plasmid or construct Host strains constructsplasmids^(a) in E. coli designation (with plasmids) designation andvarieties E. coli DH5 α MCR pVA 891 plasmid DH-pVA89 pVA 891 E. coli DH5α MCR pVA891::insert 3 LSN3 pLSN3 E. coli DH5 α MCR pVA891::insert 4LSN4 pLSN4 E. coli DH5 α MCR pVA891::insert 5 LSN5a pLSN5 E. coli DH5 αMCR pVA891::insert 6_(a-b) LSN6_(a-b) pLSN6a-c E. coli DH5 α MCRpVA891::ORF3a** DH-pVA/ORF3a** pVA-ORF3a** E. coli DH5 α MCR pET 28(+)::ORF3* DH-pET/ORF3 pET-ORF3 E. coli, BL 21 DE3 pVA 891 plasmidBL-pVA891 pVA 891 E. coli, BL 21 DE3 pLSN4 BL-pLSN4 pLSN4 E. coli, BL 21DE3 pET 28 (+)::ORF3* BL-pET/ORF3* pET-ORF3* Integrated recombinantrecovered recombinant Pneumococcal mutants plasmid^(a) mutants'designation plasmids' designation S. pneumoniae CP 1200 pVA891::insert 3SN3 pLSN3 S. pneumoniae CP 1200 pVA891::insert 4 SN4 pLSN4 S. pneumoniaeCP 1200 pVA891::insert 5 SN5 pLSN5 S. pneumoniae CP 1200 pVA891::insert6_(a-b) SN6 pLSN6_(a-c) S. pneumoniae CP 1200 pLSN4 SN4-4G pLSN4-4G S.pneumoniae CP 1200 pVA891::ORF3a** SN4-S10

EXAMPLE 2 Identification of Mutants With Altered C3-degrading Activity

[0075] Individual pneumococcal transformants were screened by ELISA fortheir altered C3 degrading activity. The pneumococcal tansformants weregrown individually in THB in the presence of erythromycin (0.05 μg/ml)in microtitre plates up to log phase and diluted 1/10 in SMP medium(0.05 μg of erythromycin/ml). The SMP bacterial cultures were grown upto log phase and incubated with C3 (0.83 μg of C3/ml of culture) for 2-4hrs. After incubation with C3, 100 μls of each individual transformantwas transferred to an ELISA binding plate and incubated overnight at 4°C. The plates were washed with PBS (10 μM phosphate buffer saline+0.05%Tween-20) three times. 100 μl of HRP-conjugated goat polyclonal antibodyspecific to human complement C3 (1:10000 dilution of 48 mg/ml) was addedto each well and the plates were incubated for 1-2 hrs at 37° C. Eachmicrotitre plate was washed with PBS as described above. 100 μl of 30%OPD (12 mg of O-Phenylenediamine (Zymed, South San Francisco, Calif.) in30 ml of Citrate buffer (200 mM Na₂HPO₄ and 100 mM citric acid-pH 5.0),and 12 μl of 30% H₂O₂) was added to each well and the plates wereincubated for 30 min in dark. The reaction was stopped by the additionof 50 μls of 2.5 M H₂SO₄ to each well. The amount of undegraded C3 leftin the samples was detected by HRP-conjugated goat polyclonal antibodyspecific to human complement C3. The assay was standardized so thatwells containing undegraded C3 had an O.D. 490=˜1.0. Wells with degradedC3 had reduced optical density readings resulting from decreased bindingof anti-C3 antibodies. The optical densities of the mutant and parentstrains were compared to that of negative controls (medium withdifferent concentrations of C3) to calculate the percent of C3 degradingactivities. There were four mutants, SN3, SN4, SN5 and SN6, withelevated C3 degrading activity (2.2 fold- Table 3) compared to theactivity of their parent strain CP1200. This finding was confirmed laterby Western immunoblotting for the pneumococcal mutant SN4. SN4-S10(disrupted CppA gene) were also mutants of CP1200 with reduced C3degrading activity. TABLE 3 ELISA results for C3 degradation by parentand hyper active mutants of pneumococcal strains strains each sampleELISA reading *Percentage of or controls at 490 nm C3 degraded in**negative control 0.608   0% CP1200 (parent) 0.30   51% SN3 (mutant)0.20 67.2% SN4 (mutant) 0.162 73.4% SN5 (mutant) 0.23 60.0% SN5 (mutant)0.23 60.0%

[0076] Immunoblotting was performed using ECL Western blottinigprotocols (Amersham Life Sciences, Arlington Heights, Ill.). Thepneumococcal mutants or E coli cultures with or without plasmids weregrown from freezer stock cultures, in THB or LB up to log phase andincubated with C3 (0.83 μg of C3/ ml) for 2-4 hrs, the cultures werespun down (2,500 rpm for 15 min RT or 4° C.) and the surernatants werecollected. The optical densities of the cultures were carefullymonitored and samples were equalized before being subjected toincubation with C3. Equal amounts of all collected supernatantscontaining undegraded C3 were applied to 7.5% or 10% SDS-PAGE gels underreducing conditions. The gel was transblotted to nitrocellulose membrane(75 volts; 4° C.) for 1 hr. Proteins were transferred in this exampleand in subsequent examples from gels to nitrocellulose membranes using aHoeffer transfer apparatus in Towbin buffer (3.03 g Tris, 14.4 g glycineand 200 ml Methanol in 1 litre volume pH.8.3; Towbin et al. (1979) PNAS:4350-4354) for 1 hr at 70 volts or gels were stained with 0.125%Coomassie Brilliant Blue R-250 (Pierce, Rockford, Ill.) made in 50%Methanol and 10% Acetic acid.

[0077] The blot was incubated in 10% skim milk (skim milk powder) for 1hr (room temperature) or overnight (4° C.) with gentle shaking. The blotwas washed in TTBS (0.1% Tween, 20 mM Tris, 137 mM Saline Buffer)several times and incubated with a 1:1000 dilution of HRP-conjugatedgoat antihuman C3, polyclonal antibody, IgG fraction (ICNPharmaceuticals/Cappel, Costa Mesa, Calif.) made in 3×TTBS+3% BSA for 1hr with gentle shaking. The incubated blot was washed again severaltimes in TTBS and incubated for one minute in chemiluminescent reagents(1:1 ratio of 2× luminol Enhancer and 2× stable peroxide solutions,Pierce, Rockford, Ill.). This blot was exposed to films for 5 sec toseveral seconds in the dark and the films were developed. The SDS-PAGEgels always contained pre-stained high molecular weight markers(Bethesda Research laboratories, life Sciences, Grand Island, N.Y.)ranging from 200 kd to 19 kd. The washes and incubations were performedat room temperature with a gentle shaking unless stated otherwise.

[0078] Electroporation of chromosomal DNA from the hyper-active.pneumococcal mutants, SN3, SN4, SN5 and SN6 into E. coli DH5 α MCRcompetent cells gave rise to E. coli transformants with rescuedrecombinant plasmids. E. coli DH5 α MCR transformants, LSN3, LSN4, LSN5.LSN6. LSN4G contained plasmids (Table 2 from pneumococcal mutants, SN3,SN4 SN5, SN6 and SN4-4G mutants respectively). Details of E. colistrains containing different constructs are listed in Table 2 (supra).Restriction analysis (Hind III) revealed that the inserts were indeedrecombinant plasmids. Different sizes of recombinant plasmids wereobtained from each hyperactive pneumococcal mutant Recombinant plasmids,pLSN3 and pLSN4 recovered from mutants SN3 and SN4 were the same size(˜7.8 kb) and their insert size was ˜2.4 kb. The size of the insert ofan ˜11 kb recombinant plasmid, pLSN5, obtained from the pneumococcalmutant SN5 was about 5.6 kb. The fourth pneumococcal mutant, SN6, gavetwo different, ˜6.5 kb and ˜10.5 kb recombinant plasmids, pLSN6_(a) andpLSN6_(b) which had inserts of 1.1 kb and 5.1 kb respectively. Thesepneumococcal mutants were also examined by southern hybridization. Thehyperactive pneumococcal mutant SN4 was chosen for further studies of C3degradation and therefore, the recombinant plasmid pLSN4 which wasrescued from the mutant SN4 was subjected to a full investigation.

[0079] Plasmid pLSN4 was used as a probe against EcoRI digestedchromosomal DNA samples of the pneumococcal mutants and this confirmedthe integration of the vector+insert (pLSN4) in the mutants SN3 and SN4.Both SN3 and SN4 hyperactive mutants included two hybridizing fragmentsof sizes ˜2.2 kb and ˜5.8 kb which were also present in parent strainCP1200. There were two other hybridizing vector/insert junctionfragments at ˜4.2 and ˜3.5 and these two together gave a total of ˜7.8kb (pLSN4 is ˜7.8 kb). These two bands were also present in the EcoRIdigested pLSN4 DNA sample. Both insert and vector had EcoRI sites andrepresented recombinant plasmid. The pattern of the other hyperactivemutants, SN5 and SN6, suggested that these mutants may have haddifferent inserts in their integrated recombinant plasmids.

[0080] The same plasmid pLSN4 was used to retransform the parentpneumococcal strain CP1200 to confirm its involvement in hyperactivity.As expected, the obtained mutant SN4-4G (Table 2) reproduced thephenotype of enhanced C3 degradation.

EXAMPLE 3 Isolation and Identification of C3-degrading Gene

[0081] Double stranded DNA sequence analysis was performed on the insertpart of the recombinant plasmid pLSN4. Since this insert was associatedwith C3 degrading hyper-activity, we expected to see insertion either inregulatory region of the corresponding gene or duplication of the gene;however, there was no indication of insertion in a regulatory region onthe basis of the protein data base search. This suggested thepossibility of gene duplication. There were three full open readingframes (ORFs) and one partial open reading frame with no significanthomology between the derived amino acid sequences of the above ORFs andthe proteins as provided in searches of GenBank, Blast and SwissProtdatabases. Preliminary data (Cathryn A S., et al, J. Inf Dis.170:600-608, 1994) suggested that the C3 degrading proteinase might becell wall associated (exported protein) and therefore, we looked for thepresence of a signal sequence, a proline rich domain or a LP×TG motif.None of the four ORFs had these sequence patterns and we chose ORF3, thelargest ORF for further analysis.

[0082] Double-stranded DNA of plasmid pLSN4a was prepared using CsClgradient/ethidium bromide isolation and used as a template.Oligonucleotide primers were synthesized using an applied Biosystems 391automated synthesizer, by Gibco BRL, or by Oligo 000M DNA synthesizer(Beckman Instruments Inc. La Brea, Calif.) Using the dideoxy chainterminator method (Sanger F., et al., Proc. Natl. Acad Sci. (USA)82:1074-1078, 1977) and employing Sequenase 2.0 (U.S. Biochem) and[α-³⁵S] dATP (Amersham life Sciences, Arlington Heights, Ill.)sequencing was done with an apparatus: 20110 Macrophor Electrophoresisunit (LKB Bromma) as indicated by Sequenase version 2.0 (Amersham lifesciences).

[0083] The insert (see FIG. 3) in the recombinant plasmid pLSN4,recovered from the hyper active pneumococcal homologous-recombinantmutant SN4 seemed to have restriction sites for Hinc II, Nru I, EcoR I,Cla I, EcoR V and Hpa I out of about 20 enzymes tested and this datacorrelated with the sequence data.

[0084] After reviewing the sequencing data, an internal fragment, 620bp, of the CppA gene (ORF3 of the insert) was generated by geneamplification (see Table 4 for primers) with overhangs containing Hind mrestriction sites. This fragment was subcloned into Hind III sites inthe vector pVA891, electroporated into E. coli and tested for thepresence of the insert. Finally, this subclone was transformed into wtCP1200 pneumococcal competent cells to inactivate the original CppA genein the wild type CP 1200.

[0085] DNA amplifications were carried out using a Hybaid Omnigenemachine with primers (see Table 4 for primers' sequences andamplification cycle conditions) complimentary to the 5′ and 3′ ends ofthe required DNA fragments. All the primers were constructed to includea restriction site on both ends. The amplification reaction (finalvolume 0.1 ml volume) utilized 10 μl of 10×vent buffer (finalconcentration, 1×contains: 10 mM KCl, 10 mM (NH₄)SO₄, 20mM Tris-HCl (pH8.8 at 25° C.), 2 mM MgSO₄, 0.1% triton X-100), 4 μls of 100 mMMgSO₄(final concentration 4 mM), 3 μl of 10 mM dNTP s (finalconcentration 300 μM), 50 ng template, 1 μM primers and 1 μl of 2000units/ml of vent polymerase (final concentration 2 units; enzyme wassupplied in 10 mM KCl, 0.1M EDTA, 10 μM Tn-HCl (pH 7.4), 1 mM DTT, 0.1%Triton X-100) in a final volume of 100 μl with water. Vent buffer, Ventpolymerase enzyme and MgSO₄ were purchased from New England Biolabs, MA,and dNTPs were bought from Gibco BRL.

[0086] Additional sequence was generated via fluorescent sequencingusing Applied Biosystems Model 373a DNA sequencer (DNA Sequencing CoreFacility, Interdisciplinary Center for Biotechnology Research (ICBR),University of Florida, Gainesville, Fla.). A Robotlo Workstation (ABICatalyst 800) and a Perkin Elmer-Cetus PEC 9600 thermocycler were usedin cycle sequencing reactions. The template, an amplified gene productthat represented the whole insert from plasmid pLSN4a, was cleaneddirectly from 0.7% agarose gel by Qiagen kit before it was used forautomated sequencing. The sequencing analysis was conducted withprograms (fasta, blast and other programs) available in the GCG softwarepackage. TABLE 4 Primers' sequences and gene amplification cycleconditions amplified gene fragment and sizes (kb) primers' sequence*pLSN4 insert (˜2.338 kb) PCR-1(LSN4a-L): CAG GAA GCT TGA TCT TGA AATTTC TAT GAC TCC (SEQ ID NO: 3) PCR-1(LSN4a-R): CGA GAA GCT TGA TCC TGTCGA AAT CAA AGC AGG ACG (SEQ ID NO: 4) *ORF3a (˜0.62 kb) Left PCR-2: CAGGAA GCT TTG AAA CAA TTT ATA (internal fragment of cppA) TTG AAA CCC (SEQID NO: 5) Right PCR-2: CGA GAA GCT TCA AGG AAG AAT TTT TCA GAC TTA GG(SEQ ID NO: 6) *ORF3 (˜0.726 kb) Left PCR-4: GGG GAA TTC CAT ATG AAT GTAAAT CAG (cppA gene) ATT GTA CGG (SEQ ID NO: 7) Right PCR-4: CGC CGC GGATCC TCA TAC TTC TTC AAA CCA CAA TTC (SEQ ID NO: 8) amplification cyclepLSN4 insert conditions (˜2.338 kb) ORF3a(˜0.62 kb) ORF3(˜0.720 kb)Denaturing 98° C., 3 min, one cycle 98° C., 3 min, one cycle 98° C., 3min, one cycle Denaturing 94° C., 30 sec 94° C., 30 sec Annealing 53°C., 30 sec {close oversize brace} 3 cycles 59° C., 30 sec {closeoversize brace} 3 cycles Extension 72° C., 45 sec 72° C., 54 secDenaturing 94° C., 30 sec 94° C., 30 sec 94° C., 30 sec Annealing 55°C., 30 sec {close oversize brace} 5 cycles 53° C., 30 sec {closeoversize brace} 5 cycles 59° C., 30 sec {close oversize brace} 5 cyclesExtension 72° C., 45 sec 72° C., 45 sec 72° C., 45 sec Finishing hold72° C., 5 min, one cycle 72° C. 5 min, one cycle 72° C. 5 min, one cycle

EXAMPLE 4 C3-degrading Protein Isolation and Studies

[0087] Log phase cultures of hyperactive mutants and their parentstrains were incubated with C3 for 2-4 hrs and the culture supernatantswere run on 7.5% SDS-Page gels under reducing conditions and werechecked for their increased C3 degrading activity by immunoblotting withHRP-conjugated polyclonal antibody to C3. This experiment demonstratedthat mutants SN4 and SN4-4G (obtained by retransformation of CP 1200with the recombinant plasmid pLSN4 rescued from SN4) were more activethan their parent strain CP 1200 in C3 degradation. Both α and β chainsC3 were almost completely degraded by the mutants after 4 hoursincubation whereas the degradation was incomplete for the parent strain.The CppA protein appeared to preferentially degrade the C3α chain.

[0088] A620 bp internal portion of the CppA gene was ligated into HindIII site of pVA 891 and the construct was transformed into CP 1200competent cells. The obtained transformant was tested for its ability todegrade C3. The ORF3 mutant was found to have a poor activity. The ∝chain of the C3 molecule was degraded and the β-chain was less degraded,by SDS-PAGE and western blotting analysis in comparison with its parentstrain CP 1200. The reduced activity rather than a complete absence ofactivity in the mutant indicated that the potential for the presence ofanother fully functional gene encoding another C3 degrading proteinasein the mutant.

[0089] The entire cppA gene was amplified and cloned into Nde I and BamH I sites of pet-28 b(+) (Novagen, INC. Madison, Wis.) and the gene wasincorporated with a His-Tag in its N-terminus region. The entire genewas positioned in the vector in frame as confirmed by sequence analysis.The plasmid construct was transformed into E. coli DH5∝ MCR strain forstabilization and the presence of the insert was verified before thevector and insert were transformed into E. coli BL 21 D3 (Novagen)protease deficient strain for expression. The colonies containing theplasmid constructs were selected on LB medium containing kanamycin (30μg/ml).

[0090] Protein was isolated according to the Pet System manual (Madison,Wis.) for small scale or large scale preparations. The BL 21 DE3 straincontaining the construct (pet 28 b(+)::ORF3 (cppA gene) was induced byIPTG and the expressed protein, CppA, was solubilized. Forsolubilization, the induced bacterial cultures were centrifuged and thepellet was resuspended in TES (50 mM Tris; 1 mM EDTA; 100 mM NaCl). Theresuspension was sonicated (6×15 sec pulses at a high output setting:about 50 watts) on ice and spun down to collect the pellet. The pelletwas washed in TES (50 mM Tris; 1 mM EDTA; 100 mM NaCl) twice and finallythe pellet was treated with 6 mM G-HCl+1 mM DTT+1% Tween-20 for 3 hrs at4° C.

[0091] The solubilized protein was diluted 1:10 in TTS (1% Tween, 50 mMTris, 0.7M NaCl) and dialyzed against TTS (1% Tween, 50 mM Tris .0.7MNaCl) to remove Guanidine-HCl, DTT and EDTA. The dialysed CppA proteinwas purified by Nickel column chromatography using the Pet system manualinstructions (Novagen, INC. Madison, Wis. Nickel column (2.5 ml) waspoured and after removal of Guanidine-HCl, DTT and EDTA, the expressedHis-Tagged CppA protein was applied to the Nickel column forpurification. The eluted fractions were tested for His-Tagged-CppAprotein by 10% SDS-PAGE gel and Coomassie Brilliant Blue R- 250staining. The protein was kept on ice at 4° C. or frozen in smallaliquots at −80° C. until required.

[0092] The CppA protein (about 600 ng per ml of the reaction mixture)was incubated with human complement C3 (0.83 μg of C3 per ml of thereaction mixture) for 4 hrs at 37° C. in the presence of PBS and anegative control without protein was simultaneously set up. The sampleswere analyzed by 7.5% or 10% SDS-PAGE gel under reducing conditions andwestem-blotting (ECL Western blotting protocols -Amersham Life Sciences,Arlington Heights, Ill.).

[0093] As described above, the PCR product of the whole ORF3 gene wassubcloned into pet vector pET28 b(+) (Novagen, Madison, Wis.) with aHis-tag in the amino terminus position and the construct was introducedinto protease deficient strain E. coli BL 21 DE3 (Table 2) after it wasstabilized in E. coli DH5α MCR. The E. coli BL 21 DE3 with the constructwas subjected to induction by IPTG. Total cell protein extracts of theinduced and uninduced cultures were tested. The expressed His-taggedORF3 protein (˜29 kd) was identified in the insoluble fraction of theinduced protein sample on 10% SDS-Page gel.

[0094] The following reagents were used for solubilization for 3 hrs at4° C. or 1 hr at room temperature: TES (50 mM Tris, 1 mM EDTA, 1M NaCl;(b) 6 mM G-HCl+1 mM DTT; (c) 6mM G-HCl+1 mM DTT+1% Tween 20; (d) 6mMG-HCl+1 nM DTT+1% Triton X-100. Both “c” and “d” treatments made theexpressed protein soluble as it wasobserved on 10% SDS-PAGE gel. Thetreatment with “c” reagent was chosen for subsequent large scalepreparations. The solubilized protein was dialysed followed bypurification through the nickel column and examined for its functionagainst C3.

[0095] For SDS-PAGE gels used in this example and above, total cellproteins or soluble or insoluble protein fractions were extractedaccording to Pet-system manual (Madison, Wis.). The proteins wereseparated by SDS-PAGE gels (7.5% or 10% or 15% resolving gel and 4.5%stacking gel) in the discontinuous system of Laemmli (Laemmli, U. K.,Nature 227:680-685,1970). Briefly, samples were combined with loadingbuffer (final concentration in samples was 7.57 mg/ml of Tris, 2% SDS,10% Glycerol and 1.25mgtml of Bromphenol blue, ±5% β-mercaptoethanol)and either boiled 5 min or loaded directly on the resolvinpg gels.Pre-stained high molecular weight standards (Protein markers (kd):lysozyme, 14,300; β-lactoglobulin, 18,400; carbonic anhydrase, 29,000;ovalbumin, 43,000; bovine serum albumin, 68,000; phophorylase B, 97,400myosin, 200,00 (Bethesda research laboratories, life sciences, GrandLand, N.Y.) were included on the gels. The large SDS-PAGE gels wereelectrophoresed at 15 mA for 14 hrs or 10 mA for 20 hr. Mini gels wereelectrophoresed around 2-3 hrs at a constant voltage (100-150 volts).

[0096] The expressed protein was incubated with C3 and the amount of C3present was assessed by Western immunoblotting.

[0097] Immunoblotting analysis suggested that the samples that containedthe expressed protein degraded C3 molecules. The undegraded C3 wasdetected by polygonal antibodies specific to human complement C3 andthis was clearly seen on the developed film in the case of the negativesample. Both ∝ and β chains of C3 molecules were seemed to besusceptible to the activity of the ORF3 protein in comparison with thenegative control which did not contain any ORF3 protein; however, the ∝chain was almost completely degraded while the β chain was partiallydegraded in the ORF3 samples.

EXAMPLE 5 Conservation of the C3-degrading Gene in Clinical Isolates

[0098] To examine the conservation of the gene cppA, an internalfragment of cppA was used as a probe to determine the presence of genecppA in EcoRI digested genomic DNA of different clinical (serotypes) ofpneumococcal isolates by southern hybridization. In the same experiment,the pneumococcal parent CP1200 and the hyperactive mutants SN4 andSN4-4G (both mutants containing the same plasmids-see Table 2) were alsoincluded to confirm the duplication of the cppA gene in the mutants.Southern hybridization was performed using non-radioactive DIG labeledinternal fragment of the gene as a probe. The clinical isolates, type1,type3, type 14F and virulent type 23F showed a hybridized band of about2.3 kb which was also present in the control pneumococcal strain CP1200and in the SN4 mutants. This common band indicates that the cppA genewas present in all isolates tested. The SN4 mutants also contained asecond band with a size of about 3.5 kb indicating the presence of agene duplication. The 3.5 kb size is consistent with the observationthat plasmid pLSN4 has two restriction endonuclease recognition sitesfor EcoRI, one in the insert region and a second in the vector. Hencethe restriction digestion with EcoRl produces two fragments of about4.175 kb ( 3.531 kb of vector+0.649 kb of insert) and 3.539 kb (˜1.67 kbform insert +about 1.869 kb from vector) from the recombinant plasmid.The cppA gene was located on the 1.67 kb portion of the insert and hencethe ˜3.539 kb restricted fragment of the recombinant plasmid containedthe cppA gene and only this band would hybridize to the probe which wasan internal fragment of the cppA gene; therefore, in the case of themutants with duplicated cppA gene, the second hybridized band at ˜3.5 kbrepresented the duplicated cppA gene.

1 9 726 base pairs nucleic acid single linear DNA (genomic) 1 ATGAATGTAAATCAGATTGT ACGGATTATT CCTACTTTAA AAGCTAATAA TAGAAAATTA 60 AATGAAACATTTTATATTGA AACCCTTGGA ATGAAGGCCT TGTTAGAAGA ATCGGCCTTT 120 CTGTCACTAGGTGACCAAAC GGGTCTTGAA AAGCTGGTTT TAGAAGAAGC TCCCAGTATG 180 CGTACTCGTAAGGTAGAGGG AAGAAAAAAA CTAGCTAGAT TGATTGTCAA GGTGGAAAAT 240 CCCTTAGAAATTGAAGGAAT CTTATCTAAA ACAGATTCGA TTCATCGATT ATATAAAGGT 300 CAAAATGGCTACGCTTTTGA AATTTTCTCA CCAGAAGATG ATTTGATTTT GATTCATGCG 360 GAAGATGACATAGCAAGTCT AGTAGAAGTA GGAGAAAAGC CTGAATTTCA AACAGATTTG 420 GCATCAATTTCTTTAAGTAA ATTTGAGATT TCTATGGAAT TACATCTCCC AACTGATATC 480 GAAAGTTTCTTGGAATCATC TGAAATTGGG GCATCCCTTG ATTTTATTCC AGCTCAGGGG 540 CAGGATTTGACTGTGGACAA TACGGTTACC TGGGACTTAT CTATGCTCAA GTTCTTGGTC 600 AATGAATTAGACATAGCAAG TCTTCGCCAG AAGTTTGAGT CTACTGAATA TTTTATTCCT 660 AAGTCTGAAAAATTCTTCCT TGGTAAAGAT AGAAATAATG TTGAATTGTG GTTTGAAGAA 720 GTATGA 726241 amino acids amino acid single linear protein 2 Met Asn Val Asn GlnIle Val Arg Ile Ile Pro Thr Leu Lys Ala Asn 1 5 10 15 Asn Arg Lys LeuAsn Glu Thr Phe Tyr Ile Glu Thr Leu Gly Met Lys 20 25 30 Ala Leu Leu GluGlu Ser Ala Phe Leu Ser Leu Gly Asp Gln Thr Gly 35 40 45 Leu Glu Lys LeuVal Leu Glu Glu Ala Pro Ser Met Arg Thr Arg Lys 50 55 60 Val Glu Gly ArgLys Lys Leu Ala Arg Leu Ile Val Lys Val Glu Asn 65 70 75 80 Pro Leu GluIle Glu Gly Ile Leu Ser Lys Thr Asp Ser Ile His Arg 85 90 95 Leu Tyr LysGly Gln Asn Gly Tyr Ala Phe Glu Ile Phe Ser Pro Glu 100 105 110 Asp AspLeu Ile Leu Ile His Ala Glu Asp Asp Ile Ala Ser Leu Val 115 120 125 GluVal Gly Glu Lys Pro Glu Phe Gln Thr Asp Leu Ala Ser Ile Ser 130 135 140Leu Ser Lys Phe Glu Ile Ser Met Glu Leu His Leu Pro Thr Asp Ile 145 150155 160 Glu Ser Phe Leu Glu Ser Ser Glu Ile Gly Ala Ser Leu Asp Phe Ile165 170 175 Pro Ala Gln Gly Gln Asp Leu Thr Val Asp Asn Thr Val Thr TrpAsp 180 185 190 Leu Ser Met Leu Lys Phe Leu Val Asn Glu Leu Asp Ile AlaSer Leu 195 200 205 Arg Gln Lys Phe Glu Ser Thr Glu Tyr Phe Ile Pro LysSer Glu Lys 210 215 220 Phe Phe Leu Gly Lys Asp Arg Asn Asn Val Glu LeuTrp Phe Glu Glu 225 230 235 240 Val 33 base pairs nucleic acid singlelinear DNA (genomic) 3 CAGGAAGCTT GATCTTGAAA TTTCTATGAC TCC 33 36 basepairs nucleic acid single linear DNA (genomic) 4 CGAGAAGCTT GATCCTGTCGAAATCAAAGC AGGACG 36 33 base pairs nucleic acid single linear DNA(genomic) 5 CAGGAAGCTT TGAAACAATT TATATTGAAA CCC 33 35 base pairsnucleic acid single linear DNA (genomic) 6 CGAGAAGCTT CAAGGAAGAATTTTTCAGAC TTAGG 35 36 base pairs nucleic acid single linear DNA(genomic) 7 GGGGAATTCC ATATGAATGT AAATCAGATT GTACGG 36 36 base pairsnucleic acid single linear DNA (genomic) 8 CGCCGCGGAT CCTCATACTTCTTCAAACCA CAATTC 36 50 base pairs nucleic acid single linear DNA(genomic) 9 GCTCCCAGTA TGCGTACTCG TAAGGTAGAG GGAAGAAAAA AACTAGCTAG 50

What is claimed is:
 1. An isolated protein compising at least an 80%sequence identity of SEQ ID NO: 2 and capable of degrading humancomplement protein C3.
 2. The protein of claim 1 wherein the protein isisolated from S. pneumoniae.
 3. The protein of claim 1 wherein theprotein binds human complement protein C3.
 4. The protein of claim 1,wherein the protein is a recombinant protein.
 5. The protein of claim 1wherein the protein is an isolated protein
 6. The protein of claim 1having a molecular weight as determined on a 10% polyacrylamide gel ofbetween about 24 kDa to about 34 kDa
 7. A peptide comprising at least 15sequential amino acids from the protein of claim
 1. 8. An isolatedprotein comprising SEQ ID NO:
 2. 9. A peptide comprising at least 15sequential amino acids from SEQ ID NO:
 2. 10. A protein comprising SEQID NO: 2, wherein the protein has a molecular weight as determined on a10% polyacrylamide gel of between about 24 kDa to about 34 kDa
 11. Theprotein of claim 10 wherein the protein is isolatable from S.pneumoniae.
 12. The protein of claim 10 wherein the protein is arecombinant protein.
 13. The protein of claim 10 wherein the proteindegrades human complement rotein C3.
 14. A protein comprising aminoacids 1-50 of SEQ ID NO:
 2. 15. A nucleic acid fragment comprisingnucleic acids 1246 to 1863 of FIG. 1A.
 16. A protein that degrdes humancomplement protein C3 and wherein nucleic acid encoding the proteinhybridizes to SEQ ID NO: 1 under hybridization conditions of 6×SSC,5×Denhardt, 0.5% SDS, and 100 μg/ml fragmented and denatured salmonsperm DNA hybridized overnight at 65° C. and washed in 2×SSC, 0.1% SDSone time at room tempe for about 10 minutes followed by one time at, 65°C. for about 15 minutes followed by at least one wash in 0.2×SSC; 0.1%SDS at room temperature for at least 3-5 minutes.
 17. An immune-systemstimulating composition comprising an effective amount of an immunesystem-stimulating peptide or polypeptide comprising at least 15 aminoacids from a protein comprising at least an 80% sequence identity withSEQ ID NO: 2 and capable of degding human complement protein C3.
 18. Thecomposition of claim 17 wherein the protein is isolatable from S.pneumoniae.
 19. The immune system stimulating composition of claim 15further comprising at least one other immune stimulating peptide,polypeptide or protein from S. pneumoniae.
 20. An antibody capable ofspificaUy binding to a protein comprising at least a 90% sequenceidentity with SEQ ID NO: 2 and capable of degrading human complementprotein C3.
 21. The antibody claim 20 wherein the antibody is amonoclonal antibody.
 22. The antibody of claim 20 wherein the antibodyis an antibody fragment
 23. The antibody of claim 20, wherein theantibody is a polyclonal antibody.
 24. The antibody of claim 20, whereinthe antibody is obtained from a mouse, a rat, human, or a rabbit
 25. Anucleic acid fragment capable of hybridizing to SEQ ID NO: 1 underhybridization conditions of 6×SSC, 5×Denhardt, 0.5% SDS, and 100 μg/mlfragmented and denatured salmon sperm DNA hybridized overnight at 65° C.and washed in 2×SSC, 0.1% SDS one time at room temperature for about 10minutes followed by one time at, 65° C. for about 15 minutes followed byat least one wash in 0.2×SSC, 0.1% SDS at room temperature for at least3-5 minutes.
 26. The nucleic acid of claim 25 isolated from an S.pneumoniae genome.
 27. The nucleic acid of claim 25 wherein the nucleicacid fragment encodes at least a portion of a protein.
 28. The nucleicacid of claim 27 wherein the protein degrades human complement C3. 29.The nucleic acid fragment of claim 27 wherein the nucleic acid fragmentencodes a protein that does not degrade human complement C3.
 30. Thenucleic acid of claim 25 in a nucleic acid vector.
 31. The nucleic acidof claim 30 wherein the vector is an expression vector capable ofproducing at least a portion of a protein.
 32. A cell comprising thenucleic acid of claim
 25. 33. The cell of claim 32 wherein the cell is abacterium or a eukaryotic cell.
 34. An isolated nucleic acid fragentcomprising the nucleic acid sequencegctcccagtatgcgtactcgtaaggtagagggaagaaaaaaactagctag.
 35. A method forproducing an immune response to S. pneumoniae in an animal comprisingthe steps of: administering a composition comprising a therapeuticallyeffective amount of at least a portion of a protein to a mammal, whereinnucleic acid encoding the protein hybridizes to SEQ ID NO: 1 underhybridization conditions of 6×SSC, 5×Denhardt, 0.5% SDS, and 100 μg/mlfragmented and denatured salmon sperm DNA, hybridized overnight at 65°C. and washed in 2×SSC, 0.1% SDS one time at room temperature for about10 minutes followed by one time at 65° C. for about 15 minutes followedby at least one wads in 0.2×SSC, 0.1% SDS at room temperature for atleast 3-5 minutes; and obtaining an immune response to the protein. 36.The method of claim 35 wherein the immune response is a B cell response.37. The method of claim 35 wherein the immune response is a T cellresponse.
 38. The method of claim 35 wherein the at least a portion of aprotein is at least 15 amino acids in length.
 39. The method of claim 35wherein the composition filrter comprises at least one other proteinfirom S. pneumoniae.
 40. The method of claim 35 wherein the proteincomprises at least 15 amino acids of SEQ ID NO:
 2. 41. A bacteriacomprising an insertional mutation, wherein the insertion mutation is ina gene encoding a protein capable of degrading human complement C3. 42.The bactera of claim 41 wherein the bacteria comprises an insertionalduplication mutation.
 43. An isolated protein of about 24 kDa to about34 kDa from Streptococcus pneumoniae that is capable of binding to anddegrading human complement C3.
 44. A method for inhibiting Streptococcuspneumoniae-mediated C3 degradation comprising the step of: contacting aStreptococcus pneumonia bacterium with antibody capable of binding to aprotein with the amino acid sequence of SEQ ID NO:
 2. 45. An isolatednucleic acid fragment comprising the nucleic acid sequence of SEQ ID NO:1
 46. An RNA fragment transcribed by a double-stranded DNA sequencecomprising SEQ ID NO:
 1. 47. The antibody of claim 20 wherein theantibody inhibits the binding of Streptococcus pneumoniae bacterium tohuman complement C3 protein.
 48. The antibody of claim 20 wherein theprotein is isolated and purified from S. pneumoniae.
 49. The antibody ofclaim 20, wherein the protein is a recombinant protein.
 50. The antibodyof claim 20, wherein the protein has a molecular weight as determined ona 10% polyacrylamide gel of between about 24 kDa to about 34 kDa.
 51. Anantibody that specifically binds to a peptide comprising at least 15sequential amino acids from a protein, wherein the protein comprises atleast an 80% sequence identity with SEQ ID NO: 2 and wherein the proteinbinds human complement protein C3.
 52. An antibody that specificallybinds to a protein comprising SEQ ID NO:
 2. 53. An antibody thatspecifically binds to a peptide comprising at least 15 sequential aminoacids from SEQ ID NO: 2, wherein said peptide binds human complement C3.54. The antibody of claim 20, wherein the protein degrades humancomplement protein C3.
 55. An antibody that specifically binds to apeptide consisting of at least 15 sequential amino acids from SEQ ID NO:2, wherein the antibody inhibits the binding of Streptococcus pneumoniaebacterium to human complement C3 protein.
 56. An antibody thatspecifically binds to a protein comprising amino acids 1-50 of SEQ IDNO:
 2. 57. An antibody that specifically binds to a protein, wherein theprotein binds human complement protein C3 and wherein nucleic acidencoding the protein hybridizes to SEQ ID NO: 1 under hybridizationconditions of 6×SSC, 5×Denhardt, 0.5% SDS, and 100 μg/ml fragmented anddenatured salmon sperm DNA hybridized overnight at 65° C. and washed in2×SSC, 0.1% SDS one time at room temperature for about 10 minutesfollowed by one time at, 65° C. for about 15 minutes followed by atleast one wash in 0.2×SSC, 0.1% SDS at room temperature for at least 3-5minutes.
 58. An antibody that specifically binds to a protein of about34 kDa from Streptococcus pneumoniae, wherein the protein binds to humancomplement C3.
 59. The antibody fragment of claim 22 wherein theantibody fragment comprises at least one variable domain.
 60. Theantibody of claim 59, wherein the variable domain is obtained from amouse, a rat, a human, or a rabbit.
 61. A chimeric protein comprising atleast one antibody variable domain, wherein the antibody variable domainspecifically binds to a protein comprising at least a 80% sequenceidentity with SEQ ID NO: 2 and wherein the protein binds humancomplement protein C3.
 62. The chimeric protein of claim 61, wherein theantibody variable domain is obtained from a mouse, a rat, a human, or arabbit.
 63. A composition comprising an antibody that specifically bindsto a protein comprising at least a 80% sequence identity with SEQ ID NO:2 and wherein the protein binds human complement protein C3.
 64. Thecomposition of claim 63 further comprising a pharmaceutically acceptablecarrier.
 65. A composition comprising an antibody that specificallybinds to a protein comprising at least a 80% sequence identity with SEQID NO: 2, wherein the protein binds human complement protein C3, whereinthe antibody inhibits the binding of Streptococcus pneumoniae bacteriumto human complement C3 protein.
 66. A composition comprising at leastone antibody variable domain, wherein the antibody variable domainspecifically binds to a protein comprising at least a 80% sequenceidentity with SEQ ID NO: 2 and wherein the protein binds humancomplement protein C3.
 67. The composition of claim 66, wherein thevariable domain is obtained from a mouse, a rat, a human, or a rabbit.68. A method for inhibiting Streptococcus pneumoniae-mediated C3binding, the method comprising contacting S. pneumoniae bacteria with anantibody that specifically binds to a protein comprising at least a 80%sequence identity with SEQ ID NO: 2 and wherein the protein binds humancompiemtent protein C3.
 69. The method of claim 68, wherein thecontacting S. pneumoniae bacteria with an antibody is by administeringthe antibody to an animal.
 70. The method of claim 69 wherein the animalsuffers from a S. pneumonia bacterial infection.
 71. The method of claim70 wherein the infection is a localized or systemic bacterial infection.72. The method of claim 70 wherein the infection is selected from thegroup consisting of bacterial pneumonia, bacterial meningitis, and anear infection.
 73. The method of claim 69 wherein the administering ofthe antibody prevents S. pneumonia bacterial colonization.
 74. A methodof augmenting S. pneumonia opsonization, the method comprisingadministering to an animal an antibody that specifically binds to aprotein comprising at least a 80% sequence identity with SEQ ID NO: 2,wherein the protein binds human complement protein C3, wherein theantibody inhibits the binding of Streptococcus pneumoniae bacterium tohuman complement C3 protein.